The AMPK phosphorylation on serines 132 and 150 of cingulin regulates its binding to α-tubulin and epithelial morphogenesis. (A) Coimmunoprecipitation of exogenously expressed wild-type and dephosphomimetic cingulin with endogenous α-tubulin. As to the relative intensity, the band of wild type (WT) was normalized to 1.0, and the results are expressed as means ± SE (error bars; n = 3). WB, Western blot; α-Tub, α-tubulin; CGN, cingulin. (B) SIM images of tubulin immunofluorescence in cingulin KD cells in which wild-type or dephosphomimetic mutants of cingulin were expressed. The relative signal intensity of immunofluorescence was quantified for α-tubulin and GFP for 10 cells. (C) Epithelial morphogenesis in 3D culture in collagen IA gel of control and cingulin KD cells with or without the expression of wild-type or dephosphomimetic cingulin. (D) Quantification of the isotropy or anisotropy of the colonies of control and cingulin KD Eph4 cells with or without the expression of wild-type or dephosphomimetic cingulin. The ratio of the shortest length (blue arrow) to that of the longest (red arrow) of the Eph4 cell colonies was determined as the isotropic index. The results are expressed as means ± SE (error bars) as quantified from three independent experiments. Ctrl, control. Bars: (B) 10 µm; (C and D) 20 µm.